Imaging the pharmacokinetics and therapeutic availability of the bispecific CD3xTRP1 antibody in syngeneic mouse tumor models

Abstract

BackgroundCD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the absolute tumor uptake and the extracellular dose. To optimize their currently limited efficacy in solid tumors, increased understanding of their pharmacokinetics and in vivo internalization is needed. MethodsHere were studied the pharmacokinetics and in vivo internalization of CD3xTRP1, a fully murine Fc-inert bsAb, in endogenous TRP1-expressing immunocompetent male C57BL/6J mice bearing TRP1-positive and negative tumors over time. Matching bsAbs lacking TRP1- or CD3-binding capacity served as controls. BsAbs were radiolabeled with 111In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging and ex vivo biodistribution analyses. Co-injection of 111In- and 125I-labeled bsAb was performed to investigate the in vivo internalization by comparing tissue concentrations of cellular residing 111In versus effluxing 125I. Anti-tumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry. ResultsSPECT/CT and biodistribution analyses showed that CD3xTRP1 specifically targeted TRP1-positive tumors and CD3-rich lymphoid organ and uptake peaked 24 hours pi (KPC3-TRP1: 37.7{+/-}5.3 %ID/g, spleen: 29.0{+/-}3.9 %ID/g). Studies with control bsAbs demonstrated that uptake of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues was primarily receptor-mediated. Together with CD3xTRP1 in the circulation being mainly unattached, this indicates that CD3+ T cells are generally not traffickers of CD3-bsAbs to the tumor. Additionally, "antigen-sink" effects by TRP1-expressing melanocytes were not observed. We further demonstrated rapid internalization of CD3xTRP1 in KPC3-TRP1 tumors (24h pi: 54.9{+/-}2.3% internalized) and CD3-rich tissues (spleen, 24h pi: 79.7{+/-}0.9% internalized). Therapeutic effects by CD3xTRP1 were observed for TRP1-positive tumors and consisted of high tumor influx of CD8+ T cells and neutrophils, which corresponded with increased necrosis and growth delay. ConclusionsWe show that CD3xTRP1 efficiently targets TRP1-positive tumors and CD3-rich tissues primarily through receptor-mediated targeting. We further demonstrate rapid receptor-mediated internalization of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues. Even though this significantly decreases the therapeutical available dose, CD3xTRP1 still induced effective anti-tumor T-cell responses and inhibited tumor growth. Together, our data on the pharmacokinetics and mechanism of action of CD3xTRP1 pave the way for further optimization of CD3-bsAb therapies. Graphical abstractImaging the pharmacokinetics and therapeutic availability of the bispecific CD3xTRPl antibody in syngeneic mouse tumor models O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/543829v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@64532aorg.highwire.dtl.DTLVardef@900338org.highwire.dtl.DTLVardef@c356f2org.highwire.dtl.DTLVardef@8165c1_HPS_FORMAT_FIGEXP M_FIG C_FIG